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Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10 −5 substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.

The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Root‐knot nematodes (RKNs), particularly Meloidogyne incognita, are among the most damaging and prevalent agricultural pathogens due to their ability to infect roots of almost all crops. The best strategy for their control is through the use of resistant cultivars. However, laborious phenotyping procedures make it difficult to assess nematode resistance in breeding programs. For common bean, this task is especially challenging because little has been done to discover resistance genes or markers to assist selection. We performed genome‐wide association studies and quantitative trait loci mapping to explore the genetic architecture and genomic regions underlying the resistance to M. incognita and to identify candidate resistance genes. Phenotypic data were collected by a high‐throughput assay, and the number of egg masses and the root‐galling index were evaluated. Complex genetic architecture and independent genomic regions were associated with each trait. Single nucleotide polymorphisms on chromosomes Pv06, Pv07, Pv08, and Pv11 were associated with the number of egg masses, and SNPs on Pv01, Pv02, Pv05, and Pv10 were associated with root‐galling. A total of 216 candidate genes were identified, including 14 resistance gene analogs and five differentially expressed in a previous RNA sequencing analysis. Histochemical analysis indicated that reactive oxygen species might play a role in the resistance response. Our findings open new perspectives to improve selection efficiency for RKN resistance, and the candidate genes are valuable targets for functional investigation and gene editing approaches. Core Ideas Genetic mapping reveals complex genetic architecture of common bean response to RKN. Root‐galling and number of egg masses might be controlled by independent genetic mechanisms. Promising candidates for functional investigation were recognized. Histochemical analysis indicated that ROS play a role in the resistance response.

Angular leaf spot (ALS) causes major yield losses in the common bean (Phaseolus vulgaris L.), an important protein source in the human diet. This study describes the saturation around a major quantitative trait locus (QTL) region, ALS10.1, controlling resistance to ALS located on linkage group Pv10 and explores the genomic context of this region using available data from the P. vulgaris genome sequence. DArT-derived markers (STS-DArT) selected by bulk segregant analysis and SCAR and SSR markers were used to increase the resolution of the QTL, reducing the confidence interval of ALS10.1 from 13.4 to 3.0 cM. The position of the SSR ATA220 coincided with the maximum LOD score of the QTL. Moreover, a new QTL (ALS10.2ᵁC) was identified at the end of the same linkage group. Sequence analysis using the P. vulgaris genome located ten SSRs and seven STS-DArT on chromosome 10 (Pv10). Coincident linkage and genome positions of five markers enabled the definition of a core region for ALS10.1 spanning 5.3 Mb. These markers are linked to putative genes related to disease resistance such as glycosyl transferase, ankyrin repeat-containing, phospholipase, and squamosa-promoter binding protein. Synteny analysis between ALS10.1 markers and the genome of soybean suggested a dynamic evolution of this locus in the common bean. The present study resulted in the identification of new candidate genes and markers closely linked to a major ALS disease resistance QTL, which can be used in marker-assisted selection, fine mapping and positional QTL cloning.

Silicon (Si) efficiently controls Podosphaera xanthii in melon by affecting epidemic components related to the infection and colonization processes. Root amendments of Si prime melon defences, but its mode of action when sprayed on the leaves is unknown. The effects of potassium silicate (PS) on the development of the pathogen were observed by scanning electron microscopy. Both forms of PS application reduced conidial germination and delayed colony development. At 120 h after inoculation (hai), differentiation of the conidiophores was inhibited only by the root treatment and at 144 hai visual differences between PS treatments accentuated. Comparison of foliar PS with compounds of contrasting pH and ionic strength (KOH, KCl and PEG) showed that only PS and PEG inhibited conidial germination ruling out these variables as inhibitory factors. Alteration of the leaf surface tension was also excluded since it was affected by all compounds. The results indicate that foliar-applied PS affects P. xanthii conidial germination of and colony development by modifying the osmotic potential of the leaf surface.

Background Sporisorium scitamineum is the causal agent of sugarcane smut, affecting global sugarcane production. Despite advances in smut genomics, the relationships between fungal genetic diversity, host adaptation, and virulence remain elusive. Methods We applied chromosome-level genome sequencing (Oxford Nanopore and Illumina technologies) of two haploid strains ( MAT-1 x MAT-2 ) per isolate and high-depth transcriptomic profiling (Illumina) during early infection (48 hpi) of a more virulent isolate (SSC04) in resistant (SP80-3280) and susceptible (IAC66-6) sugarcane genotypes. Results Despite the overall genomic similarity (99.9%), we identified nine highly polymorphic genomic islands (HPIs). The most variant HPI harbors the mating-type loci, where dense sequence variation, intrachromosomal rearrangements, and inversions, potentially linked to transposable element remnants, were observed. Additionally, the genome-wide analysis revealed non-synonymous single-nucleotide variants (SNVs) in 160 genes, including those involved in vesicular trafficking and candidate-secreted effectors. Transcriptomic profiling of the more virulent isolated revealed host-dependent transcriptional reprogramming in response to immune and metabolic cues, driving distinct infection strategies: in resistant plants, the fungus upregulated genes associated with detoxification, nitrogen starvation responses, and cell wall-degrading enzymes, while in susceptible hosts, it induced genes related to hyphal growth, lipid catabolism, and the unfolded protein response. The repertoire of expressed candidate effector genes also varied according to host and isolate genotypes. Conclusions These findings uncover genomic signatures and context-dependent transcriptional regulation shaping the adaptive landscape of S. scitamineum virulence, identifying targets for pathogen monitoring and breeding for resistance.

The fungus Podosphaera xanthii affects melon crops and presents several races controlled by race-specific resistance genes. The accession PI 414723 is resistant to races 1, 3 and 5 and it is a suitable source of resistance genes. The inheritance of resistance to these races was analyzed on 87 F2 plants from the cross of PI 414723 × Védrantais, and resistance to all three races could be explained by the segregation of a single dominant gene, although a digenic model could also be accepted. A genetic map was assembled with 206 markers, and co-segregation analysis of resistance phenotypes indicated the existence of two linked loci in linkage group II, one conferring resistance to races 1 and 5 (denominated Pm-x1,5), and the second to race 3 (denominated Pm-x3), located 5.1 cM apart. This study reports for the first time the existence of Pm-x3 and the genetic locations of these resistance genes from PI 414723. O fungo Podosphaera xanthii afeta a cultura do meloeiro, apresenta diversas raças e é controlado por genes de resistência raça-específica. O acesso PI414723 é resistente às raças 1, 3 e 5 e é uma fonte adequada de genes de resistência. A herança da resistência a essas raças foi analisada em 87 plantas F2 do cruzamento PI414723 × Védrantais. A resistência às três raças pode ser explicada pela segregação de um gene dominante, embora um modelo digênico também possa ser aceito. Um mapa genético foi obtido com 206 marcadores e análise de co-segregação de fenótipos resistentes indicou a existência de dois locos ligados, posicionados no grupo de ligação II, um conferindo resistência às raças 1 e 5 (denominado Pm-x1,5) e o segundo à raça 3 (denominado Pm-x3), localizado a 5,1cM. Este estudo relata pela primeira vez a existência de Pm-x3 e as localizações genéticas desses genes de resistência de PI414723.

Key message Successful orange rust development on sugarcane can potentially be explained as suppression of the plant immune system by the pathogen or delayed plant signaling to trigger defense responses. Puccinia kuehnii is an obligate biotrophic fungus that infects sugarcane leaves causing a disease called orange rust. It spread out to other countries resulting in reduction of crop yield since its first outbreak. One of the knowledge gaps of that pathosystem is to understand the molecular mechanisms altered in susceptible plants by this biotic stress. Here, we investigated the changes in temporal expression of transcripts in pathways associated with the immune system. To achieve this purpose, we used RNA-Seq to analyze infected leaf samples collected at five time points after inoculation. Differential expression analyses of adjacent time points revealed substantial changes at 12, 48 h after inoculation and 12 days after inoculation, coinciding with the events of spore germination, haustoria post-penetration and post-sporulation, respectively. During the first 24 h, a lack of transcripts involved with resistance mechanisms was revealed by underrepresentation of hypersensitive and defense response related genes. However, two days after inoculation, upregulation of genes involved with immune response regulation provided evidence of some potential defense response. Events related to biotic stress responses were predominantly downregulated in the initial time points, but expression was later restored to basal levels. Genes involved in carbohydrate metabolism showed evidence of repression followed by upregulation, possibly to ensure the pathogen nutritional requirements were met. Our results support the hypothesis that P. kuehnii initially suppressed sugarcane genes involved in plant defense systems. Late overexpression of specific regulatory pathways also suggests the possibility of an inefficient recognition system by a susceptible sugarcane genotype.

Abstract Leaf scald is a destructive sugarcane disease caused by the bacterium Xanthomonas albilineans (Ashby) Dowson. This pathogen presents the gene cluster SPI-1 T3SS, a conserved feature in pathogens vectored by animals. In this study, the competence of Mahanarva fimbriolata (Stål), a spittlebug commonly found in sugarcane fields in Brazil, was evaluated for the transmission of X. albilineans. Artificial probing assays were conducted to investigate the ability of M. fimbriolata adults to acquire X. albilineans from artificial diets containing the pathogen with subsequent inoculation of X. albilineans into pathogen-free diets. Plant probing assays with M. fimbriolata adults were conducted to evaluate the acquisition of X. albilineans from diseased source plants and subsequent inoculation of healthy recipient sugarcane plants. The presence of X. albilineans DNA in saliva/diet mixtures of the artificial probing assays and both insects and plants of the plant probing assays were checked using TaqMan assays. The artificial probing assays showed that M. fimbriolata adults were able to acquire and inoculate X. albilineans in diets. Plant probing assays confirmed the competence of M. fimbriolata to transmit X. albilineans to sugarcane. Over the entire experiment, 42% of the insects had acquired the pathogen and successful inoculation of the pathogen occurred in 18% of the recipient-susceptible sugarcane plants at 72 or 96 h of inoculation access period. Assays evidenced the vector competence of M. fimbriolata for transmission of X. albilineans, opening new pathways for investigating the biology and the economic impacts of the interaction between X. albilineans and M. fimbriolata.

Generation means was used to study the mode of inheritance of resistance to anthracnose stalk rot in tropical maize. Each population was comprised of six generations in two trials under a randomized block design. Inoculations were performed using a suspension of 10(5) conidia mL-1 applied into the stalk. Internal lesion length was directly measured by opening the stalk thirty days after inoculation. Results indicated contrasting modes of inheritance. In one population, dominant gene effects predominated. Besides, additive x dominant and additive x additive interactions were also found. Intermediate values of heritability indicated a complex resistance inheritance probably conditioned by several genes of small effects. An additive-dominant genetic model sufficed to explain the variation in the second population, where additive gene effects predominated. Few genes of major effects control disease resistance in this cross. Heterosis widely differed between populations, which can be attributed to the genetic background of the parental resistant lines. Análise de médias foi usada para estudar o modo de herança da resistência a antracnose do colmo em milho tropical. Cada população foi composta de seis gerações que foram avaliadas para resistência em dois ensaios no delineamento de blocos ao acaso. As inoculações foram realizadas usando uma suspensão de 10(5) conídios mL-1 aplicados dentro do colmo. O comprimento interno de lesão foi medido diretamente após abertura do colmo 30 dias após a inoculação. Os resultados indicaram modos contrastantes de herança. Em uma população, os efeitos gênicos dominantes predominaram. Embora as interações, aditivo x dominante e aditivo x aditivo, também foram encontradas. Valores intermediários de herdabilidade indicaram herança da resistência complexa, provavelmente condicionada por vários genes de pequeno efeito. O modelo genético aditivo-dominante foi suficiente para explicar a variação na segunda população, onde os efeitos aditivos predominaram. Poucos genes de grande efeito controlam a resistência neste cruzamento. Heterose diferiu amplamente entre as populações o que pode ser explicado pelo background genético das linhagens genitoras resistentes.